Acrylamide, N,N′-methylenebisAcrylamide (MBAA), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), 3-amino-1,2,4-triazole (3-AT), l -histidine, adenine sulfate, sulforhodamine B, bisphenol A ethoxylate diacrylate (BPA) (512 g/mol), and poly(ethylene glycol) diacrylate (PEG-DA) (700 g/mol) were purchased from Sigma-Aldrich. The photoinitiator Irgacure 369 (I-369) was donated by BASF Corporation. Methacryloxyethyl thiocarbonyl rhodamine B (PolyFluor 570) was purchased from Polysciences. Commercial yeast (S. cerevisiae, active dry yeast, Fleischmann’s) was purchased from Tom Thumb (Richardson, TX). Yeast extract, yeast nitrogen base without amino acids, peptone, d -(+)-glucose, d -histidine, and trypan blue were purchased from Fisher Scientific. TPM [3-(trimethoxysilyl) propyl methacrylate] was purchased from Acros Organics. Rain-X was purchased from Wal-Mart (Richardson, TX). All chemicals were used as received without further purification.
Adenine sulfate
Manufactured by Merck Group
Adenine sulfate is a chemical compound commonly used in laboratory settings. It serves as a precursor for the synthesis of various biomolecules and is a key component in cellular processes. The compound has a molecular formula of (NH2)2C5H4N5·H2SO4 and is a white crystalline powder.
Automatically generated - may contain errors
Lab products found in correlation
Ade2 y, by Integrated DNA Technologies (2 mentions)
Pmel13, by Integrated DNA Technologies (2 mentions)
Sanger sequencing, by Eurofins (2 mentions)
N n methylenebisacrylamide, by Merck Group (1 mentions)
3 amino 1 2 4 triazole 3 at, by Merck Group (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Acrylamide, by Merck Group (1 mentions)
Ammonium persulfate, by Merck Group (1 mentions)
Sulforhodamine b, by Merck Group (1 mentions)
L histidine, by Merck Group (1 mentions)
Poly ethylene glycol diacrylate pegda, by Merck Group (1 mentions)
Methacryloxyethyl thiocarbonyl rhodamine b polyfluor 570, by Polysciences (1 mentions)
Irgacure 369, by BASF (1 mentions)
3 protocols using adenine sulfate
1
Hydrogel Microparticle Synthesis Protocol
2
Constructing S. cerevisiae ade2::TdFBA1 Strains
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S. cerevisiae strains in which the coding region (ORF) of T. delbrueckii FBA1 was integrated into the S. cerevisiae ADE2 gene, in opposite orientation to ADE2 so that it is not functional, were constructed using CRISPR-Cas9 as follows. The ADE2-targeting sgRNA ADE2.Y from DiCarlo et al. (2013) (link) was synthesized as a gene fragment by Integrated DNA Technologies, and inserted into the sgRNA plasmid pMEL13 from Mans et al. (2015) (link) by restriction digestion and ligation. This plasmid was then transformed into S. cerevisiae strain IMX585 expressing Cas9 (Mans et al., 2015 (link)), together with a repair template containing the T. delbrueckii FBA1 ORF (TdFBA1-S or TdFBA1-R allele) flanked with homology to S. cerevisiae ADE2 in reverse orientation (bases 564456..564832 and 565952..566366 of S. cerevisiae chromosome XV). Sequences of the ade2::TdFBA1-S and ade2::TdFBA1-R constructs are given in Supplementary file 3 . Transformants were selected on YPAD (YPD (Formedium) supplemented with 40 μg/ml adenine sulfate (Sigma)) containing 200 μg/ml G418. ADE2 knockouts were identified by formation of red colonies. Successful integrants were confirmed by PCR amplification of the ade2::TdFBA1 locus and Sanger sequencing (Eurofins). Two replicate ade2::TdFBA1-S strains were designated C1 and C4, and two replicate ade2::TdFBA1-R strains were designated L1 and L3.
3
CRISPR-Cas9-Mediated Integration of Heterologous FBA1 in Yeast
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S. cerevisiae strains in which the coding region (ORF) of T. delbrueckii FBA1 was integrated into the S. cerevisiae ADE2 gene, in opposite orientation to ADE2 so that it is not transcribed, were constructed using CRISPR-Cas9 as follows. The ADE2-targeting sgRNA ADE2.Y from DiCarlo et al. ( 2013) was synthesized as a gene fragment by Integrated DNA Technologies, and inserted into the sgRNA plasmid pMEL13 from Mans et al. (2015) by restriction digestion and ligation. This plasmid was then transformed into S. cerevisiae strain IMX585 expressing Cas9 (Mans et al., 2015) , together with a repair template containing the T. delbrueckii FBA1 ORF (TdFBA1-S or TdFBA1-R allele) flanked with homology to S. cerevisiae ADE2 in reverse orientation (bases 564456..564832 and 565952..566366 of S. cerevisiae chromosome XV). Transformants were selected on YPAD (YPD (Formedium) supplemented with 40 μg/ml adenine sulfate (Sigma)) containing 200 μg/ml G418. ADE2 knockouts were identified by formation of red colonies. Successful integrants were confirmed by PCR amplification of the ade2::TdFBA1 locus and Sanger sequencing (Eurofins). Two replicate ade2::TdFBA1-S strains were designated C1 and C4, and two replicate ade2::TdFBA1-R strains were designated L1 and L3. Sequences of the ade2::TdFBA1-S and ade2::TdFBA1-R constructs are given in Supplementary File S1.
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